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Overview
• Resolving power and how to measure it
• Why two-dimensional LC?
• Setup of a 2D-LC System
• Different modes of 2D-LC
• Heart-Cutting 2D-LC
• Multiple Heart-Cutting 2D-LC
• High Resolution Sampling 2D-LC
• Comprehensive 2D-LC
• One software for all 2D-LC modes
• Online 2DLC for Biopharmaceuticals – 4 case studies
September 23, 2016
2
Increasing Lab Efficiency UHPLC and Other Solutions
• Accelerating Analysis
• UHPLC particles (sub-2µm columns + UHPLC systems)
• Automation
• Automated sample preparation
• Valve solutions
• Increasing peak capacity
• Longer columns
• Online 2D-LC
Why Two-Dimensional LC? General Thoughts in Separation Science
“Resolving power is what it is all
about in analytical separation
science.“
• Increased peak capacity
• Further resolution of a complex mixture that cannot be separated on a single
column
• Sample cleanup by removing matrix or interfering compounds
• Increase sample throughput (two separations going on at once)
Why Two-Dimensional LC?
September 23, 2016
5
Heart-cutting 2D-LC
Hidden impurity?
Multiple heart-cutting
2D-LC
Co-elution?
Comprehensive 2D-LC
More resolving power for a
comprehensive analysis?
2D LC Modules 1. Dimension
2. Dimension
1290 Infinity
Binary Pump
1290 Infinity
Binary Pump
1290 Infinity
Autosampler or 1260
HiP Autosampler
Optional
1260/1290
Infinity
Detector
One or two
1290 Infinity
TCC
Optional
1260/1290
Infinity
Detector
1260/1290
Infinity
detector
1260 Infinity Binary
or Quaternary Pump
1260 Infinity
Capillary Pump 1260 Infinity
Autosampler
For 1st
dimension
chromatogram
and peak-
triggering
To monitor
waste-line
Almost any Agilent pump or autosampler in the 1st dimension!
Almost any detectors are supported!
A 1290 Infinity Binary Pump for the 2nd dimension is required.
2D-LC System Configuration
September 23, 2016
8
1D Pump Autosampler 1D Column
2D Pump
2D Column
Injector
2D Detector
1D Detector
(optional)
Consider Dedicated Bio-inert HPLC Protein Analysis for First Dimension
Unspecific surface binding
Low throughput, low resolution
Peak tailing for critical proteins
Corrosion and pH issues with standard LC
Decreased column lifetime and chromatographic performance
Metal-free chromatography needs (e.g. Cr Speciation)
High pH applications
Phosphorylated compounds, oligonucleotides
September 23, 2016
10
Agilent 1260 Infinity II Bio-Inert Quaternary LC The New Standard in Bioanalysis
September 23, 2016
11
100% Bio-inert Precious sample does not touch metal
surfaces
pH range 1-13 (shortterm 14)
2 M salt, 8 M urea
No stainless steel in mobile phase flow
path
New capillary technology
UHPLC capability 600 bar
Ease of Use and Robustness Corrosion resistant
Active seal wash
Quaternary buffer mixing
Bio-HPLC columns for biotherapeutic
characterization
The choice for both, bioanalytical and biopurification up to 10 ml/min
Bio-Inert Components
Inert materials: Titanium (mechanical parts), PEEK, Ceramics, PTFE, FFKM
Needle
SST Housing
for Robustness
PEEK and
ceramic for metal free
sample path
Seat
PEEK Seat (inside)
SST Holder and Screw
for Robustness
Metal-Clad
PEEK capillary
New Capillary Design Ensures Bio-Inertness
September 23, 2016
13
Capillaries Metal-clad PEEK capillary (Titanium Capillary also available)
Newest fitting design: Hybrid technique
New capillary technolgy
enables 600 bar AND is completely metal free !!
Quaternary Buffer Mixing with Buffer Advisor Use with Ion Exchange Chromatography
September 23, 2016
14
Most intuitive software to set
up and edit methods within
seconds:
• 1st dimension gradient
• 2nd dimension gradient
• Gradient shift
• Time-segments
• Method parameter
• Method set-up calculator
• Reference chromatogram
overlay for heart-cutting
• Time-based or peak-based
mode
2D-LC Acquisition Software One easy-to-use software for all operation modes
15
September 23, 2016
Data Analysis (Multiple) Heart-Cutting and High Resolution Sampling
September 23, 2016
16
The 2D-LC Heart-Cut Viewer allows straightforward data analysis of
(multiple) heart-cutting and High resolution sampling 2D-LC data.
2D-LC Major Types of Operation
September 23, 2016
17
(MULTIPLE)
HEART-CUTTING 2D-LC
COMPREHENSIVE 2D-LC
2D-LC – Heart-Cutting
Heart-cutting 2D-LC (LC-LC):
LC1
LC2
Parts of the 1D effluent are injected onto
2D system
Long 2D gradients possible good data
quality
Limited 2D information
18
September 23, 2016
Limitation of Heart-Cutting 2D-LC
19
September 23, 2016
20min
4.5min
Peaks lost while second dimension is still busy with analysis
1D
2D 4.5min
From Single to Multiple Heart-Cutting 2D-LC
September 23, 2016
20
20min
1D
2D 4.5min
4.5min
4.5min 4.5min 4.5min
Idea: De-couple the first and second dimensions by intermediately storing peaks
eluted from the first dimension for later analysis in the second dimension.
Multiple Heart-Cutting 2D-LC
September 23, 2016
21
LC 1
LC 2
Valve and loop configuration
• Several parts of the 1D
effluent are injected onto 2D
system
• Long 2D gradients possible,
therefore, high resolution in
2D possible
• 2D information for multiple
1D peaks
High-Resolution Sampling 2D-LC
September 23, 2016
22
LC 1
LC 2
Valve and loop configuration
• Multiple consecutive heart-cuts
across a 1D peak or region of interest
• Long 2D gradients possible
• Detailed 2D information for a certain
1D peak
• Quantification of compounds
coeluting in 1D
Comprehensive 2D-LC
LC1
LC2
1st peak from
1st dimension
2nd peak from
1st dimension
3rd peak from
1st dimension
The whole 1D effluent is injected onto
2D system
(Ultra)Short 2D gradients necessary Good data
quality with fast pumps & detector
Full („comprehensive“) 2D information!
23
September 23, 2016
LC1
LC2
min
sec
Comprehensive 2D-LC How it works
September 23, 2016
24
Loop 1
Loop 2
Loop 1
Loop 2
• Collection of effluent
from the first dimension
column in loop 1
• Analysis of the content
from loop 2
• Collection of effluent
from the first dimension
column in loop 2
• Analysis of the content
from loop 1
Applications of Comprehensive 2D-LC
Analysis of very complex samples
Traditional Chinese Medicine (TCM)/Chinese Herbal Medicine (CHM):
• Holistic healthcare system
• Pharmaceutical efficacy regarded to depend on synergistic effects of multiple
components of the plants
Biopharmaceutical analysis:
• Peptide maps
• Complex glycan pattern
September 23, 2016
25
Finger Printing and Profiling Analysis
Case Studies Online 2DLC of Monoclonal Antibodies
• Monoclonal Antibody Digests HILIC × RPLC-MS
Focus: Peak capacity
• Characterization of Monoclonal Antibodies Protein A x WCX
Focus: Combination of two different workflows
• Characterization of Monoclonal Antibodies SEC x WCX
Focus: Combination of two different workflows
• Characterization of Charge Variants WCX x RP
Focus: Desalting after IEX prior to MS
Case Study 1 Comprehensive 2D-LC
Orthogonal Separations of mAb Tryptic Digest
• Aggregation Studies
• Charge Variant Analysis
• Peptide Mapping
• Glycan Profiling
• Titer Analysis
• And others…
Analysis of Monoclonal Antibody Digests with the Agilent 1290 Infinity 2D-LC Solution
Focus: High Peak Capacity
Comprehensive 2D-LC
LC1
LC2
1st peak from
1st dimension
2nd peak from
1st dimension
3rd peak from
1st dimension
The whole 1D effluent is injected onto
2D system
(Ultra)Short 2D gradients necessary Good data
quality with fast pumps & detector
Full („comprehensive“) 2D information!
32
September 23, 2016
LC1
LC2
min
sec
Comprehensive 2D-LC mAb Tryptic Digest Summary
• Higher peak capacity with 2D-LC
• Improved separation using othogonal column combination: HILIC x RP
• Proof of concept with the clear differentiation of stressed and non-stressed
antibody samples
• Ideal combination of Agilent 1290 Infinity 2D-LC Solution coupled to an Agilent
6530 Accurate-Mass Q-TOF LC/MS
Case Study 2 Combining Two Techniques
Protein A and WCX
• Aggregation Studies
• Charge Variant Analysis
• Peptide Mapping
• Glycan Profiling
• Titer Analysis
• And others…
Combination of Two Analyses Offline Approach
min 1 2 3 4 5 6
mAU
0
100
200
300
400
500
Fraction collection after first dimension
min 2 4 6 8 10 12
mAU
0
25
50
75
100
125
150
175 Offline charge
variant analysis in
the second
dimension
Requires hands-on time!
Characterization of Monoclonal Antibodies Protein A and Weak Cation Exchange Chromatography
Focus: Combination of two important Quality Attribute Analyses
High Resolution Multiple Heart Cuts
2D-LC with High Resolution Sampling Protein A Titer Analysis (First Dimension)
Consecutive Cuts Across the Peak
Precision of Retention Time and Area
1D mAb peak after protein A
monolith
RSD RT [%] RSD Area [%]
0.08 0.8
Peak # RSD RT [%] RSD Area
[%]
1 0.22 1.11
2 0.24 1.48
3 0.21 1.20
High Resolution Multiple Heart Cuts Protein A - WCX Summary
• Fully automated combination of two different quality attribute analyses
Protein A titer analysis and charge variants
• No hands-on time required between the two analyses
• High precision of retention time and area found in both dimensions
• Also suitable for process control samples
Characterization of Monoclonal Antibodies Size Exclusion and Weak Cation Exchange Chromatography
Focus: Combination of two important Quality Attribute Analyses
Case Study 3 Combining Two Techniques
SEC and WCX
• Aggregation Studies
• Charge Variant Analysis
• Peptide Mapping
• Glycan Profiling
• Titer Analysis
• And others…
Multiple Heart-Cutting 2D-LC
September 23, 2016
47
LC 1
LC 2
Valve and loop configuration
• Several parts of the 1D
effluent are injected onto 2D
system
• Long 2D gradients possible,
therefore, high resolution in
2D possible
• 2D information for multiple
1D peaks
Aggregation Analysis of mAb
SEC in the First Dimension
min 2 4 6 8 10 12
mAU
0
100
200
300
400
500
min 1 2 3 4 5 6 7 8
mAU
0
2
4
6
8
10
12
14
Zoom
Ag
gre
ga
te P
ea
k
Ma
in P
ea
k -
Mo
no
me
r
2D LC Multiple Heart Cut SEC – WCX Precision of Retention Time and Area
Peak # RSD RT
[%]
RSD
Area [%]
Acidic 1 0.08 1.28
Acidic 2 0.07 2.09
Main 0.04 0.36
Basic 0.06 1.08
Peak # RSD RT [%] RSD Area
[%]
Aggregate 0.07 0.45
Main 0.02 0.26
Case Study 3 Combining SEC and WCX Summary
• Fully automated combination of two different quality attribute analyses
Size Exclusion and Charge Variants
• No hands-on time required between the two analyses
• High precision of retention time and area found in both dimensions
Case Study 4 Charge Variants Analysis LCMS Ion Exchange and Reversed-Phase Chromatography
Focus: 2D-LC as desalting step after IEX
Charge profiles of Rituximab Innovator and Biosimilar
min 5 10 15 20 25 30 35
mAU
0
200
400
600
800
1000
Rituximab Biosimilar
Rituximab Innovator
Online MS detection Intact Mass Analysis of Biosimilar Rituximab – 3 peaks
same mass as innovator peak
+128 Da
+128 Da
Case Study 4 Charge Variants Analysis LCMS Summary
• Innovator and Biosimilar Rituximab charge variants were analyzed using
weak cation exchange chromatography
• Online 2DLC-MS qualification was enabled by automated desalting and
denaturation
• The three peaks of the biosimilar vs one peak of the innovator were
qualified as c-terminal lysine variants (mass shift of 128 Da)
Further information can be found…
• Schneider, S. 2D-LC/MS Characterization of Charge Variants Using Ion Exchange and
Reversed-Phase Chromatography, Agilent Technologies Application Note, publication
number 5991-6673EN, 2016.
• Schneider, S. Online 2D-LC Characterization of Monoclonal Antibodies Using Protein A and
Weak Cation Exchange Chromatography, Agilent Technologies Application Note, publication
number 5991-6848EN, 2016.
• Schneider, S. Online 2D-LC Characterization of Monoclonal Antibodies with Size Exclusion
and Weak Cation Exchange Chromatography, Agilent Technologies Application Note,
publication number 5991-6906EN, 2016.
• Vanhoenacker, G et al.; Analysis of Monoclonal Antibody Digests with the Agilent 1290
Infinity 2D-LC Solution Part 2: HILIC × RPLC-MS, Agilent Technologies Application Note,
publication number 5991-4530EN, 2014.
• Schneider, S; Naegele, E; Krieger, S. Online 2D-LC Analysis of Complex N-Glycans in
Biopharmaceuticals Using the Agilent 1290 Infi nity 2D-LC Solution Agilent Technologies
Application Note, publication number 5991-5349EN, 2015.